blood products advisory committee meeting april 26, 2007 ben marchlewicz, ph.d. prism r&d,...
DESCRIPTION
Abbott’s Strategy in Blood Screening for Chagas Disease Develop a recombinant antigen based assay for antibodies to T. cruzi on the PRISM platform Well-characterized antigens Presenting multiple epitopes Non-biohazardous Develop a recombinant antigen based Confirmatory assay for resolution of PRISM Repeat Reactive specimens.TRANSCRIPT
Blood Products Advisory Committee Meeting
April 26, 2007
Ben Marchlewicz, Ph.D.PRISM R&D, Program Manager
Abbott’s approach to Chagas Testing PRISM Chagas
Description of the assayPerformance
Chagas ConfirmatoryDescription of the assayPerformance
Abbott’s Strategy in Blood Screening for Chagas Disease
Develop a recombinant antigen based assay for antibodies to T. cruzi on the PRISM platform
Well-characterized antigens Presenting multiple epitopes Non-biohazardous
Develop a recombinant antigen based Confirmatory assay for resolution of PRISM Repeat Reactive specimens.
PRISM Chagas Assay
PRISM Chagas Screening Assay Design Configuration
+ * * *
* *
Anti- T. cruzi Ab in Sample
rAg-Coated P
*
ACR-Mouse Anti-Human IgG
Conjugate
H2O2
Light
TcF coated P
FP Co-coated P
+ Transfer
Wash
Conjugate Wash
Dual rAg Coated P, Single mAb Conjugate, Two–Step Assay
Multi-epitope rAg Amino acids Mol. Wt. (Kda)
TcF 85 8.3
FP3 693 76
FP6 994 110
FP10 722 78
Multi-epitope Recombinant Antigens used in PRISM Chagas Assay
Specificity
Study Total Repeatedly Reactive
Confirmed Positive
Resolved Specificity
In-House Feasibility
Testing7,755 4 1 99.96%
Field Study 4,100 8 3 99.88%
Total 11,855 12 4 99.93%
Random Donors
Specificity
Study Total Specificity
Leishmania visceral(sourced from India)
30 100%
Leishmania cutaneous(sourced from India)
11 100%
Malaria 10 100%
Cross Reactivity with other Parasitic Infections
PRISM Chagas Assay SensitivityChagas Positive Specimens (confirmed by RIPA)
RIPA Positive
RIPA Negative
PRISM Positive 419 0
PRISM Negative 0 0
Sensitivity
100% Sensitivity
Chagas Confirmatory Assay
Design of Chagas Confirmatory Immunostrip
Anti-hIgG (Sample Addition CTL)
hIgG-H CTL
hIgG-L CTL (Cut-off)
4 rAgs for anti-T. cruzi detection
TcF FP3 FP6 FP10
ID#
Test Bands On-board CTL bands
IMMUNOSTRIP HUMAN SPECIMEN 2 hr Incubation
Wash
Wash
1 hr Incubation ANTI-HUMAN AP CONJUGATE
10 min IncubationBCIP-NBTCOLOR
Chagas Confirmatory Assay Schematic
On-BoardControls
ChagasAntigens
Immunostrip
ID#
Visual Reading of Test Bands
Intensity Score
Absent - Faint or < on-board hIgG-L CTL +/-
= on-board hIgG-L CTL 1+ Between on-board hIgG-L CTL and hIgG-H CTL 2+
= on-board hIgG-H CTL 3+ > on-board hIgG-H CTL 4+
Sensitivity
RIPA Positive
RIPA Negative
Immunoblot Chagas
Confirmatory Positive
410* 0
Immunoblot Chagas
Confirmatory Negative
0 0
100% Sensitivity
* Also reactive in PRISM Assay & 2 reference ELISA’s
Country of Origin # of SamplesArgentina 41
Bolivia 53
Brazil 176
Chile 5
Colombia 1
Ecuador 1
El Salvador 12
Honduras 4
Mexico 33
Nicaragua 17
Surinam 1
USA 4
Venezuela 21
Unknown 41
Chagas Specimens Panel n = 410 (All are confirmed RIPA positive)
Specificity
RIPA Positive
PRISMChagas Assay Non-reactive
Immunoblot Chagas
Confirmatory Positive
1* 0
Immunoblot Chagas
Confirmatory Negative
0 500
100% Resolved Specificity
Unscreened Random Blood Donors
* Also reactive in PRISM Assay & 2 reference ELISA’s
Specificity
RIPA Positive
PRISMChagas Assay Non-reactive
Immunoblot Chagas
Confirmatory Positive
2* 0
Immunoblot Chagas
Confirmatory Negative
0 316
100% Resolved Specificity
Unrelated Medical Conditions
* Also reactive in PRISM Assay & 2 reference ELISA’s
Specificity
Study Total Specificity
Leishmania visceral(sourced from India)
30 100%
Leishmania cutaneous(sourced from India)
11 100%
Malaria 10 100%
Cross Reactivity with other Parasitic Infections
Summary
A fully automated blood screening assay for antibodies to T. Cruzi, on the PRISM system, is under development; Along with an Immunoblot Confirmatory assay, that can be performed on site, to confirm Repeat Reactive specimens to aid in donor management.